Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for detection and characterization of a wide variety of molecules and ions. This is largely due to the extremely sensitive, fast and selective detection provided by mass spectrometric methods. While mass spectrometry provides a highly effective means of identifying a wide class of molecules, its use for analyzing high molecular weight compounds is hindered by problems related to generating, transmitting and detecting gas phase analyte ions of these species.
First, analysis of important biological compounds, such as oligonucleotides and oligopetides, by mass spectrometric methods is severely limited by practical difficulties related to low sample volatility and undesirable fragmentation during vaporization and ionization processes. Importantly, such fragmentation prevents identification of labile, non-covalently bound aggregates of biomolecules, such as protein-protein complexes and protein-DNA complexes, that play an important role in many biological systems including signal transduction pathways, gene regulation and transcriptional control. Second, many important biological application require ultra-high detection sensitivity and resolution that is currently unattainable using conventional mass spectrometric techniques. As a result of these fundamental limitations, the potential for quantitative analysis of samples containing biopolymers remains largely unrealized.
For example, the analysis of complex mixtures of oligonucleotides produced in enzymatic DNA sequencing reactions is currently dominated by time-consuming and labor-intensive electrophoresis techniques that may be complicated by secondary structure. The primary limitation hindering the application mass spectrometry to the field of DNA sequencing is the limited mass range accessible for the analysis of nucleic acids. This limited mass range may be characterized as a decrease in resolution and sensitivity with an increase in ion mass. Specifically, detection sensitivity on the order of 10−15 moles (or 6×108 molecules) is required in order for mass spectrometric analysis to be competitive with electrophoresis methods and detection sensitivity on the order of 10−18 moles (or 6×105 molecules) is preferable. Higher resolution is needed to resolve and correctly identify the DNA fragments in pooled mixtures particularly those resulting from Sanger sequencing reactions.
In addition to DNA sequencing applications, current mass spectrometric techniques lack the ultra high sensitivity required for many other important biomedical applications. For example, the sensitivity needed for single cell analysis of protein expression and post-translational modification patterns via mass spectrometric analysis is simply not currently available. Further, such applications of mass spectrometric analysis necessarily require cumbersome and complex separation procedures prior to mass analysis.
The ability to selectively and sensitively detect components of complex mixtures of biological compounds via mass spectrometry would tremendously aid the advancement of several important fields of scientific research. First, advances in the characterization and detection of samples containing mixtures of oligonucleotides by mass spectrometry would improve the accuracy, speed and reproducibility of DNA sequencing methodologies. In addition, such advances would eliminate problematic interferences arising from secondary structure. Further, enhanced capability for the analysis of complex protein mixtures and multi-subunit protein complexes would revolutionize the use of mass spectrometry in the field of proteomics. Important applications include: protein identification, relative quantification of protein expression levels, identification of protein post-translational modifications, and the analysis of labile protein complexes and aggregates. Finally, advances in mass spectrometric analysis of samples containing complex mixtures of biomolecules would also provide the simultaneous characterization of both high molecular weight and low molecular weight compounds. Detection and characterization of low molecular weight compounds, such as glucose, ATP, NADH, GHT, would aid considerably in elucidating the role of these molecules in regulating a myriad of important cellular processes.
Mass spectrometric analysis involves three fundamental processes: (1) desorption and ionization of a given analyte species to generate a gas phase ion, (2) transmission of the gas phase ion to an analysis region and (3) mass analysis and detection. Although these processes are conceptually distinct, in practice each step is highly interrelated and interdependent. For example, desorption and ionization methods employed to generate gas phase analyte ions significantly influence the transmission and detection efficiencies achievable in mass spectrometry. Significantly, all ion sources currently available for preparation of gas phase ions from large biomolecules result in large ion losses during transmission and mass analysis process. Accordingly, a great deal of research is currently directed at developing new ion sources that provide improved transmission of gas phase analyte ions into conventional devices for analysis and detection.
Conventional ion preparation methods for mass spectrometric analysis have proven unsuitable for high molecular compounds. Vaporization by sublimation or thermal desorption is unfeasible for many high molecular weight species, such as biopolymers, because these compounds tend to have negligibly low vapor pressures. Ionization methods based on the desorption process, however, have proven more effective in generating ions from thermally labile, nonvolatile compounds. Such methods primarily consist of processes that initiate the direct emission of analyte ions from solid or liquid surfaces. Although conventional ion desorption methods, such as plasma desorption, laser desorption, fast particle bombardment and thermospray ionization, are more applicable to nonvolatile compounds, these methods have substantial problems associated with ion fragmentation and low ionization efficiencies for compounds with molecular masses greater than about 2000 Daltons.
To enhance the applicability of mass spectrometry for the analysis of samples containing large molecular weight species, two new ion preparation methods recently emerged: (1) matrix assisted laser desorption and ionization (MALDI) and (2) electrospray ionization (ESI). These methods have profoundly expanded the role of mass spectrometry for the analysis of high molecular weight compounds, such as biomolecules, by providing high ionization efficiency (ionization efficiency=ions formed/molecules consumed in analysis) applicable to a wide range of compounds with molecular weights exceeding 100,000 Daltons. In addition, MALDI and ESI are characterized as “soft” desorption and ionization techniques because they are able to both desorb into the gas phase and ionize biomolecules with substantially less fragmentation than conventional ion desorption methods. Karas et. al, Anal. Chem., 60, 2299-2306 (1988) and Karas et. al, Int. J. Mass Spectrom. Ion Proc., 78, 53-68 (1987) describe the application of MALDI as an ion source for mass spectrometry. Fenn, et. al, Science, 246, 64-71 (1989) describes the application of ESI as an ion source for mass spectrometry.
In MALDI mass spectrometry, the analyte of interest is co-crystallized with a small organic compound present in high molar excess relative to the analyte, called the matrix. The MALDI sample, containing analyte incorporated into the organic matrix, is irradiated by a short (≈10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. The rapid absorption of energy by the matrix causes it to desorb into the gas phase, carrying a portion of the analyte molecules with it. Gas phase proton transfer reactions ionize the analyte molecules within the resultant gas phase plume. Generally, these gas phase proton transfer reactions generate analyte ions in singly and/or doubly charged states. Upon formation, the ions in the source region are accelerated by a high potential electric field, which imparts equal kinetic energy to each ion. Eventually, the ions are conducted through an electric field-free flight tube where they are separated by mass according to their kinetic energies and are detected.
Although MALDI is able to generate gas phase analyte ions from very high molecular weight compounds (>2000 Daltons), certain aspects of this ion preparation method limit its utility in analyzing complex mixtures of biomolecules. First, fragmentation of analyte molecules during vaporization and ionization gives rise to very complex mass spectra of parent and fragment peaks that are difficult to assign to individual components of a complex mixture. Second, the sensitivity of the technique is dramatically affected by sample preparation methodology and the surface and bulk characteristics of the site irradiated by the laser. As a result, MALDI analysis yields little quantitative information pertaining to the concentrations of the materials analyzed. Finally, the ions generated by MALDI possess a very wide distribution of trajectories due to the laser desorption process, subsequent ion-ion charge repulsion in the plume and collisions with background matrix molecules. This spread in analyte ion trajectories substantially decreases ion transmission efficiencies achievable because only ions translating parallel to the centerline of the mass spectrometer are able to reach the mass analysis region and be detected.
In contrast to MALDI, ESI is a field desorption ionization method that provides a highly reproducible and continuous stream of analyte ions. It is currently believed that the field desorption occurs by a mechanism involving strong electric fields generated at the surface of a charged substrate which extract solute analyte ions from solution into the gas phase. Specifically, in ESI mass spectrometry a solution containing solvent and analyte is passed through a capillary orifice and directed at an opposing plate held near ground. The capillary is maintained at a substantial electric potential (approximately 4 kV) relative to the opposing plate, which serves as the counter electrode. This potential difference generates an intense electric field at the capillary tip, which draws some free ions in the exposed solution to the surface. The electrohydrodynamics of the charged liquid surface causes it to form a cone, referred to as a “Taylor cone.” A thin filament of solution extends from this cone until it breaks up into droplets, which carry excess charge on their surface. The result is a stream of small, highly charged droplets that migrate toward the grounded plate. Facilitated by heat and/or the flow of dry bath gases, solvent from the droplets evaporates and the physical size of the droplets decreases to a point where the force due to repulsion of the like charges contained on the surface overcomes the surface tension causing the droplets to fission into “daughter droplets.” This fissioning process may repeat several times depending on the initial size of the parent droplet. Eventually, daughter droplets are formed with a radius of curvature small enough that the electric field at their surface is large enough to desorb analyte species existing as ions in solution. Polar analyte species may also undergo desorption and ionization during electrospray by associating with cations and anions in the liquid sample.
Because ESI generates a highly reproducible stream of gas phase analyte ions directly from a solution containing analyte ions, without the need for complex, off-line sample preparation, it has considerable advantages over analogous MALDI techniques. Certain aspects of ESI, however, currently prevent this ion generating method from achieving its full potential in the analysis complex mixtures of biomolecules. First, ions generated in ESI invariably possess a wide distribution of multiply charged states for each analyte discharged because the ionization process proceeds via the formation of highly charged liquid droplets. Accordingly, ESI-MS spectra of mixtures are typically a complex amalgamation of peaks attributable to a large number of populated charged states for every analyte present in the sample. These spectra often possess too many overlapping peaks to permit effective discrimination and identification of the various components of a complex mixture. In addition, highly charged gas phase ions are often unstable and fragment prior to detection, which further increases the complexity of ESI-MS spectra.
Second, a large percentage of ions formed by electrospray ionization are lost during transmission into and through the mass analyzer. Many of these losses can be attributed to divergence in the stream of ions generated. Mutual charge repulsion of ions is a major contributor to beam spreading. In this process, charged droplets and gas phase ions formed by ESI mutually repel each other during transmission from the source to an analysis and detection region. This mutual charge repulsion significantly widens the spatial distribution of the droplet and/or gas phase ion stream and causes significant deviation from the centerline of the mass spectrometer. As the sensitivity of the ESI-MS technique depends strongly on the efficiency with which analyte ions are transported into and through a mass analyzer, the spread in gas phase ion trajectories substantially decreases detection sensitivity attainable in ESI-MS. In addition, spread in ion position is also detrimental to the resolution of the mass determination. For example, in pulsed orthogonal time-of-flight detection, the spread in ion position prior to orthogonal extraction substantially influences the resolution attainable. Divergence of the gas phase ion stream is a major source of deviations in ion start position and, hence, degrades the resolution attainable in the time-of-flight analysis of ions generated by ESI. Typically, small entrances apertures for orthogonal extraction are employed to compensate for these deviations, which ultimately result in a substantial decrease in detection sensitivity.
Finally, ESI, as a continuous ionization source, is not directly compatible with time-of-flight mass analysis. Time-of-flight (TOF) detection is currently the most widely employed detection method for large biomolecules due to its ability to characterize the mass to charge ratio of very high molecular weight compounds. To obtain the benefits from both ESI ion generation and TOF mass analysis, techniques have been developed to segment the continuous ion stream generated in ESI into discrete packets. For example, in conventional TOF analysis electrospray-generated ions are periodically pulsed into an electric field-free-flight tube positioned orthogonal to the axis along which the ions are generated. In the flight tube, the analyte ions are separate by mass according to their kinetic energies and are detected at the end of the flight tube. In this configuration it is essential that the accelerated packets of ions are sufficiently temporally separated with adequate spacing to avoid overlap of consecutive mass spectra. Although ions are generated continuously in ESI-TOF, mass analysis by orthogonal extraction is limited by the duty cycle of the extraction pulse. Most ESI-TOF instruments have a duty cycle between 5% and 50%, depending on the m/z range of the ions being analyzed. Therefore, the majority of ions formed in ESI-TOF are never actually mass analyzed or detected because ion production is not synchronized with detection.
Recently, research efforts have been directed at developing new field desorption ion sources that provide more efficient transmission and detection of the ions generated. One method of improving the transmission and detection efficiencies of ions generated by field desorption involves employing pulsed charged droplet sources that are capable of generating a stream of discrete, single droplets or droplet packets with directed momentum. As the droplets generated by such a droplet source are temporally and spatially separated, mutual charge repulsion between droplets is minimized. Further, ion formation and detection processes may be synchronized by employing a pulsed source, which eliminates the dependence of detection efficiency on the duty cycle of orthogonal extraction in time-of-flight detection.
Hager et. al reported a mass spectrum of dodecyldiamine (Molecular Mass=201 amu) by incorporating a pulsed droplet source with a Sciex TAGA 6000E mass spectrometer [Hager, D. B. et. al, Appl. Spectrosc., 46, 1460-1463 (1992)]. Using a piezoelectric source, they reported generation of a continuous stream of neutral droplets. After formation, the droplets were charged using an external charging element comprising a corona discharge positioned near the droplet stream. While Hager et al. report successful ion generation via field desorption of droplets generated by a piezoelectric source, electric fields generated by the external corona discharge were observed to significantly perturbed the trajectories of the charged droplets generated. Specifically, FIG. 3 of this reference indicates that the corona discharged caused deflection of droplet trajectories up to approximately 45° from the original trajectory of the droplet. Accordingly, Hager et al. report decreases in ion intensities by a factor of 2-3 relative to conventional electrospray ionization. Further, Hager et al. report no results with higher molecular weight species. Finally, the apparatus described by Hager et al. is not amenable to single droplet production or discretely controlled droplet formation because it employs a continuous droplet source which utilizes Rayleigh breakup of a liquid jet that in not capable of discrete pulsed droplet generation.
Feng et al. recently reported the combination of a droplet on demand piezoelectric dispenser with an electrodynamic trap to provide a pulsed source of gas phase ions [Feng et al., J. Am. Soc. Mass Spectrom., 11, 393-399 (2000)]. The electrodynamic trap consisted of two ring electrodes to which an RF voltage signal was applied between the electrodes to counter the downward force on the droplet due to gravity. Droplets were generated by a pulsed piezoelectric dispenser and charged with an external induction electrode. The authors report a 100% efficiency in capturing discrete droplets generated by the pulsed piezoelectric dispenser. The droplets remained in the electrodynamic trap until they were evaporated and/or desolvated to induce droplet fission. The droplet itself and daughter droplets, which formed during desolvation, were reported to exit the trap vertically through the upper electrode and were subsequently detected by a channel electron multiplier housed in a vacuum chamber. While Feng et al. were able to direct the exit of the parent and daughter droplets out of the electrodynamic trap, they report very poor ion transfer efficiency to the vacuum chamber. The decreased ion transfer efficiency was likely due to divergence of charged droplets upon leaving the droplet trap from the selected droplet trajectory. Feng et al. report no results with high molecular weight compounds or any applications of their ion source involving mass analysis.
Another approach to increase gas phase ion transmission and detection efficiencies involves reducing ion beam divergence using external devices to collimate charged droplets and gas phase ions formed by field desorption methods. Electrostatic ion lenses are routinely used to minimize ion beam divergence. While electrostatic ion lens may be employed to collimate or focus a diverging ion beam, most lens systems exhibit aberrations, which minimize the optimum focus conditions to a narrow mass to charge ratio (m/z) window over a limited energy range. In addition, ions that are brought to a focus via an electrostatic lens quickly diverge once past the focal point and, thus, ultimately may not be transmitted and detected.
Lui et al. describe an aerodynamic lens system that is capable of concentrating suspended particles around a central axis without the use of electrostatic lenses [Lui et al., Aerosol Science and Technology, 22, 293-313 (1995), Lui et al., Aerosol Science and Technology, 22, 314-324 (1995)]. Specifically, the authors report the used of an aerodynamic lens systems to transport droplets and particles from an intermediate pressure region (0.01-0.1 Torr) into a region of high vacuum (approximately 1×10−5 Torr) that utilizes a flow of background gas to focus in place of electric potentials. Utilizing a stream of polydispersed NaCl particles with diameters less than 0.2 μm produced by atomization, Lui et al. report greater than 90% transport efficiency to a high vacuum detection region, particle beam diameters ranging from 0.7 to 3.0 mm and particle velocities ranging from 60 to 200 meters per second. Lui et al. do not, however, describe use of an aerodynamic lens system in field desorption ion sources. Additionally, the authors do not report use of the aerodynamic lens system for sampling in mass analysis.
It will be appreciated from the foregoing that a need exists for field desorption ion sources that are capable of generating a stream of single gas phase ions or discrete, packets of gas phase ions having reduced divergence and improved spatial uniformity. The present invention provides a gas phase ion sources able to provide controlled, production of gas phase ions or discrete packets of gas phase ions, from chemical species, including high molecular weight biopolymers, with directed momentum along an ion production axis. Further, this invention describes methods and devices of determining the identity and concentration of chemical species in liquid samples using this gas phase ion source in combination with charged particle analysis.